Molecular determinants of myeloma bone disease and uses thereof

ABSTRACT

To identify molecular determinants of lytic bone disease in multiple myeloma, the expression profiles of ˜12,000 genes in CD138-enriched plasma cells from newly diagnosed multiple myeloma patients exhibiting no radiological evidence of lytic lesions (n=28) were compared to those with ≧3 lytic lesions (n=47). Two secreted WNT signaling antagonists, soluble frizzled related protein 3 (SFRP-3/FRZB) and the human homologue of Dickkopf-1 (DKK1), were expressed in 40 of 47 with lytic bone lesions, but only 16 of 28 lacking bone lesions (P&lt;0.05). DKK1 and FRZB were not expressed in plasma cells from 45 normal bone marrow donors or 10 Waldenstrom&#39;s macroglobulinemia, a related plasma cells malignancy that lacks bone disease. These data indicate that these factors are important mediators of multiple myeloma bone disease, and inhibitors of these proteins may be used to block bone disease.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit of provisional patent applicationU.S. Serial No. 60/431,040, filed Dec. 5, 2002, now abandoned.

FEDERAL FUNDING LEGEND

[0002] This invention was created, in part, using funds from the federalgovernment under National Cancer Institute grants CA55819 and CA97513.Consequently, the U.S. government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to the study of multiplemyeloma. More specifically, the present invention relates to theidentification of molecular determinants of myeloma bone disease throughcomparative global gene expression profiling.

[0005] 2. Description of the Related Art

[0006] Multiple myeloma (MM) is a rare, yet incurable malignancy ofterminally differentiated plasma cells (PC) that affects approximately15,000 persons per year in the United States, and represents the secondmost common hematopoietic malignancy. Multiple myeloma represents 13% ofall lymphoid malignancies in the white population and 31% of lymphoidmalignancies in the black population. The malignant plasma cells home toand expand in the bone marrow causing anemia and immunosuppression dueto loss of normal hematopoiesis.

[0007] Multiple myeloma is also associated with systemic oteoporosis andlocal bone destruction leading to debilitating bone pain andsusceptibility to fractures, spinal cord compression and hypercalcemia.Myeloma is the only hematological malignancy consistently associatedwith lytic bone disease and local bone destruction is limited to areasadjacent to plasma cells, suggesting that the malignant plasma cellssecrete factors that enhance osteoclast function and/or osteoblastanergy. The prevalence of bone disease varies with the presentation ofmyeloma, from smoldering myeloma, often without bone involvement, tosolitary plasmacytoma, to diffused or focal multiple myeloma wheresystemic losses of bone mineral density or focal lytic bone lesions areseen in approximately 80% of patients.

[0008] In recent years, it has become evident that lytic bone disease isnot only a consequence of myeloma, but that it is intricately involvedin promoting disease progression. Change in bone turnover rates predictsclinical progression from monoclonal gammopathy of undeterminedsignificance (MGUS) to overt myeloma by up to 3 years. While initiallyosteoclast and osteoblast activity are coupled, the coupling is lostwith disease progression. Osteoclast activity remains increased andosteoblast activity is diminished, with lytic bone disease as theconsequence. Studies in the 5T2 murine myeloma and the SCID-hu model forprimary human myeloma demonstrated that inhibition of osteoclastactivity is associated with inhibition of myeloma growth and reductionof myeloma tumor burden. These studies support reports that inhibitionof bone resorption with bisphosphonates had an anti-myeloma effect.

[0009] Whereas the biology of osteoclasts in myeloma-associated lyticbone disease has been investigated intensively, little is known aboutthe disease-associated changes in osteoblast activity and theirunderlying mechanisms. It has been suggested that in myeloma, theability of mesenchymal stem cells to differentiate into the osteogeniclineage is impaired. However, the mechanisms responsible for suchimpairment have not been elucidated.

[0010] It has been shown that comparative global gene expressionprofiling (GEP) of bone marrow plasma cells from normal healthy donorsand malignant bone marrow plasma cells from newly diagnosed multiplemyeloma represented a powerful technique for identifying candidatedisease genes and disrupted pathways involved in malignanttransformation of multiple myeloma (Zhan et al., 2002).

[0011] The prior art is deficient in a comparative analysis to identifygenes expressed in the malignant plasma cells that may be contributoryto multiple myeloma bone diseases as well as methods to diagnose andtreat multiple myeloma bone diseases. The present invention fulfillsthis longstanding need and desire in the art.

SUMMARY OF THE INVENTION

[0012] To identify the molecular determinants of lytic bone disease, theexpression profiles of ˜12,000 genes in CD138-enriched plasma cells fromnewly diagnosed multiple myeloma exhibiting no radiological evidence oflytic lesions (n=28) were compared to those with ≧3 lytic lesions(n=47). Consistent with a critical role of WNT signaling in osteoblastdifferentiation, two secreted WNT signaling antagonists, solublefrizzled related protein 3 (SFRP-3/FRZB) and the human homologue ofDickkopf-1 (DKK-1), were expressed in 40 of 47 with lytic bone lesions,but only 16 of 28 lacking bone lesions (P<0.05). Immunohistochemistryshowed high levels of DKK-1 and FRZB in plasma cells from cases withhigh gene expression. Importantly, DKK-1 and FRZB were not expressed inplasma cells from 45 normal bone marrow donors or 10 Waldenstrom'smacroglobulinemia, a related plasma cells malignancy that lacks bonedisease.

[0013] Serum derived from multiple myeloma patients with high DKK-1blocked both Wnt signaling and osteoblast differentiation in vitro.Importantly, pre-incubation of the serum with DKK-1 and FRZB antibodiesinhibited this function. Consistent with a key role for JUN incontrolling DKK-1 expression and in turn apoptosis, plasma cells derivedfrom extramedullary disease as well as primary refractory disease hadlow expression of JUN and DKK-1.

[0014] Multiple myeloma plasma cells showed a massive up-regulation ofDKK-1 and FRZB gene expression after in vivo treatment. DKK-1 and FRZBcan be upregulated in multiple myeloma plasma cells after treatment ofpatients with genotoxic drugs used to treat the disease, thus furtheringa role for DKK-1 in multiple myeloma cell apoptosis. Primary multiplemyeloma cells co-cultured with in vitro derived osteoclasts (OC) lackedapoptosis and that this was tightly correlated with the down-regulationof JUN, FOS, FOSB, and DKK-1.

[0015] Results disclosed in the present invention indicate that blockingthe production and/or secretion of DKK-1 and FRZB may prevent or reversebone loss in multiple myeloma patients. Further applications may includeusing DKK-1 and FRZB inhibitors to prevent bone loss in the generalpopulation. Additionally, Wnt signaling has recently been shown to becritical for the self renewal capacity of hematopoietic stem cells.Futhermore, a bone marrow niche required for HSC proliferation is formedby mature osteoblasts. The block to Wnt signaling by DKK1 and FRZB coulddirectly and indirectly impair hepatic stellate cell (HSC) proliferationand thus may partly account for the immunosuppression and anemia seen inmultiple myeloma. Thus blocking DKK1 and/or FRZB may also prevent orreverse the defect in hematopoeisis seen in most patients with myeloma.

[0016] Other and further aspects, features, and advantages of thepresent invention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] So that the matter in which the above-recited features,advantages and objects of the invention as well as others which willbecome clear are attained and can be understood in detail, moreparticular descriptions and certain embodiments of the invention brieflysummarized above are illustrated in the appended drawings. Thesedrawings form a part of the specification. It is to be noted, however,that the appended drawings illustrate preferred embodiments of theinvention and therefore are not to be considered limiting in theirscope.

[0018]FIGS. 1A and 1B show global gene expression patterns reflectingbone lesions in myeloma. FIG. 1A shows clusterview of normalizedexpression levels of 57 genes identified by logistic regression analysisas being significantly differentially expressed in malignant plasmacells from patients with no (n=36) and 1+MRI focal lesions (n=137)(P<0.0001). The 28 genes exhibiting elevated expression in plasma cellsfrom patients with 1+MRI lesions are ordered from top to bottom based onrank of significance. Likewise the 30 genes showing significantelevation in patients with no MRI-lesions are ordered from bottom to topbased on significance rank. Gene symbols (Affymetrix probe setidentifiers when the gene is unnamed) are listed to the left. Normalizedexpression scales range from −30 (blue) to +30 (red) as indicated belowthe data display. The four genes remaining significant after permutationadjustment are underlined.

[0019]FIG. 1B shows a bar graph of DKK1 gene expression in plasma cellsfrom normal bone marrow (BPC), patients with monoclonal gammopathy ofundetermined significance (MGUS), Waldenström's macroglobulinemia (WM),and multiple myeloma (MM) presented on the x-axis. MM samples are brokendown into three bone lesion groups: no MRI/no x-ray lesions, 1+MRI/nox-ray lesions, and 1+MRI/1+x-ray lesions. The Affymetrix Signal, aquantitative measure of gene expression derived from MAS 5.01, isindicated on the y-axis. DKK1 gene expression level in each sample isindicated by a bar, with the height of the bar proportional to geneexpression intensity. Samples are ordered from the lowest to highestDKK1 gene expression from left to right on the x-axis. The number ofsamples in each group is indicated below each group designator.Statistics for comparisons between the MM subgroups are indicated in thetext.

[0020]FIG. 2 shows RHAMM was up-regulated in multiple myeloma patientswith bone lesions.

[0021]FIG. 3 shows RHAMM rarely present in normal plasma cells andmonoclonal gammopathy of undetermined significance (MGUS), but it waspresent in virtually all human myeloma cell lines.

[0022]FIG. 4 shows securin was up-regulated in multiple myeloma patientswith bone disease.

[0023]FIG. 5 shows MIP-1α and CCR1 were “spike” genes in multiplemyeloma, but they were not correlated with lytic lesions. Black bar:CCR1; gray bar: MIP-1α.

[0024]FIG. 6 shows MIP-1α was expressed at low level in normal plasmacells (PC).

[0025]FIG. 7 shows the expression of WNT antagonist DKK-1 in multiplemyeloma with bone lesions.

[0026]FIG. 8 shows the expression of WNT decoy receptor FRZB in multiplemyeloma with lytic bone lesions.

[0027]FIG. 9 shows the expression of DKK-1 and FRZB in multiple myelomawith lytic bone lesions. Black bar: DKK-1; gray bar: FRZB.

[0028]FIG. 10 shows FRZB was expressed in tonsil plasma cells. PBC, TBC,tonsil B cells; TPC, tonsil plasma cells; BPC, bone marrow plasma cells;WPC, WBC, CLL.

[0029]FIG. 11 shows DKK-1 was not expressed in normal B cells or plasmacells. PBC, TBC, tonsil B cells; TPC, tonsil plasma cells; BPC, bonemarrow plasma cells; WPC, WBC, CLL.

[0030]FIG. 12 shows DKK-1 expression in monoclonal gammopathy ofundetermined significance (MGUS) was low relative to smoldering multiplemyeloma (SMM) and newly diagnosed multiple myeloma (MM).

[0031]FIG. 13 shows FRZB was elevated in monoclonal gammopathy ofundetermined significance (MGUS), and had higher expression insmoldering multiple myeloma (SMM) and newly diagnosed multiple myeloma(MM).

[0032]FIG. 14 shows the expression of DKK-1 and FRZB in monoclonalgammopathy of undetermined significance (MGUS) and smoldering multiplemyeloma (SMM).

[0033]FIG. 15 shows low expression of DKK-1 in extramedullary disease.

[0034]FIG. 16 shows the expression of DKK-1 and FRZB tend to be higherin plasma cells from medullary PCT than those from iliac crest. PCT,FNA.

[0035]FIG. 17 shows the expression of DKK-1 and FRZB in fine needleaspirates of medullary PCT.

[0036]FIG. 18 shows high expression of DKK-1 and FRZB in medullaryplasmacytoma.

[0037]FIG. 19 shows higher expression of DKK-1 in multiple myeloma withosteopenia.

[0038]FIG. 20 shows DKK-1 was not expressed in plasma cells fromWaldenstrom's macroglobulinemia.

[0039]FIG. 21 shows WNTSA was elevated in newly diagnosed multiplemyeloma.

[0040]FIG. 22 shows WNT5A tends to be higher in multiple myeloma withlytic lesions.

[0041]FIG. 23 shows WNTSA was also elevated in monoclonal gammopathy ofundetermined significance (MGUS) and smoldering multiple myeloma (SMM).

[0042]FIG. 24 shows WNT10B tends to be lower in multiple myeloma withlytic lesions.

[0043]FIG. 25 shows WNTSA and WNT10B tend to be inversely correlated.Black bar: WNT10B; gray bar: WNT5A.

[0044]FIG. 26 shows DKK-1 was present in an SK-LMS cell line.

[0045]FIG. 27 shows primary multiple myeloma synthesized DKK-1 protein.

[0046]FIG. 28 shows low DKK-1 expression in relapsed and primaryrefractory multiple myeloma.

[0047]FIG. 29 shows endothelin receptor B was a “spike” gene in onethird of newly diagnosed multiple myeloma.

[0048]FIG. 30 shows the expression of endothelin receptor B inmonoclonal gammopathy of undetermined significance (MGUS) and smolderingmultiple myeloma. Normal plasma cells do not express endothelin receptorB.

[0049]FIG. 31 shows the involvement of endothelin receptor B in boneformation.

[0050]FIG. 32 shows DKK-1 expression after treatment with PS-341.

[0051]FIG. 33 shows DKK-1 expression after treatment with thalomid innewly diagnosed multiple myeloma.

[0052]FIG. 34 shows DKK-1 expression after treatment with IMiD.

[0053]FIG. 35 shows DKK-1 expression after treatment with dexamethsonein newly diagnosed multiple myeloma.

[0054]FIG. 36 shows downregulation of JUN and FOS in multiple myelomacells after co-culture with osteoclasts.

[0055]FIG. 37 shows JUN & DKK-1 downregulation in osteoclast co-culture.

[0056]FIG. 38 shows WNT signaling in multiple myeloma bone disease.

[0057]FIG. 39 shows overexpression of DKK1 in low grade myeloma with theloss of expression with disease progression. Expression of DKK1 wasexamined by immunohistochemistry of myeloma bone marrow biopsies. Serialsections (550× magnification) of bone marrow biopsies from myelomapatients with high (a-b) and low (c-d) DKK1 gene expression arepresented. Slides are stained with H&E (a and c) or anti-DKK1 andsecondary antibody (b and d). Use of secondary alone failed to stainedcells (data not shown). Magnified images (1,200× magnification) arelocated in the upper left corner of each H&E image. Image a shows amyeloma with an interstitial pattern of involvement with plasma cellsexhibiting low grade morphology with abundant cytoplasm and no apparentnucleoli. Image b reveals positive staining of plasma cells in ainterstitial pattern with anti-DKK1 antibody that was greatest adjacentto bone. Image c shows a myeloma with nodular or alliterative patternwith plasma cells exhibiting high grade morphology with enlarged nucleiand prominent nucleoli. Image d reveals no positive staining of plasmawith anti-DKK1 antibody.

[0058]FIGS. 40A and 40B show DKK1 protein in the bone marrow plasma ishighly correlated with DKK1 gene expression and the presence of bonelesions. FIG. 40A shows the expression of DKK1 mRNA was detected bymicroarray and DKK1 protein by ELISA in a total of 107 cases of newlydiagnosed myeloma. Results of both assays were transformed by the logbase 2 and normalized to give a mean of 0 and variance of 1. Each barindicates the relative relationship of gene expression and proteinexpression in each sample. There was a significant correlation betweenDKK1 mRNA in myeloma plasma cells and protein in bone marrow plasma(r=0.65, P<0.001). FIG. 40B shows bar view of DKK1 protein levels inbone marrow plasma plasma cells from normal donors (BPC), patients withmonoclonal gammopathy of undetermined significance (MGUS), Waldenström'smacroglobulinemia (WM), and multiple myeloma (MM) are presented on thex-axis. MM samples are broken down into three bone lesion groups: noMRI/no x-ray lesions, 1+MRI/no x-ray lesions, and 1+MRI/1+x-ray lesions.The DKK1 protein concentration (ng/ml) is indicated on the y-axis. Toenable comparisons of DKK1 protein levels in the lower ranges, 200 ng/mlwas made the maximum value. This resulted in the truncation of a singlesample with DKK1 concentration of 476 ng/ml. DKK1 protein level in eachsample is indicated by a bar, with the height of the bar proportional toDKK1 protein levels. Samples are ordered from the lowest to highest DKK1protein levels from left to right on the x-axis. The number of samplesin each group is indicated below each group.

[0059]FIGS. 41A and 41B show recombinant DKK1 and MM plasma can blockalkaline phosphatase production in BMP-2 treated C2C12 cells in aDKK1-dependent manner. FIG. 41A shows alkaline phosphatase levels, amarker of osteoblast differentiation (y-axis) were measured in C2C12cells after 5 days of culture in the presence of 5 percent fetal calfserum alone or with BMP2, BMP2+DKK1, BMP2+DKK1+anti-DKK1, orBMP-2+DKK1+polyclonal IgG. Each bar represents the mean (±SEM) oftriplicate experiments. Note that activity of alkaline phosphataseincreased in the presence of BMP-2 and significant reduction of thisprotein by co-incubation with recombinant DKK1. Also note that anti-DKK1antibody, but not polyclonal IgG can block the repressive activity ofDKK1. FIG. 41B shows alkaline phosphatase levels (y-axis) were tested inC2C12 cells after culturing these cells for 5 days in 5 percent fetalcalf serum alone or 50 ng/ml BMP-2+10 percent normal bone marrow plasma(NS) or BMP-2+10 percent myeloma bone marrow plasma from 10 patientswith newly diagnosed myeloma (sample identified provided), or BMP2+10percent myeloma patient plasma +anti-DKK1 or goat polyclonal IgG. Eachbar represents the mean (±SEM) of triplicate experiments. DKK1concentration from each bone marrow plasma samples was determined byELISA and final concentrations in culture after 1:10 dilution areindicated on the x-axis. Note that samples with >12 ng/ml DKK1 had aneffect on alkaline phosphatase production. A star indicates P<0.05 incomparison to alkaline phosphatase in BMP2+10 percent normal human bonemarrow plasma.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention demonstrates that the secreted WNTsignaling antagonists DKK-1 and FRZB mediate bone destruction seen inmultiple myeloma. Together with emerging evidence of an absoluterequirement of Wnt signaling in osteoblast growth and differentiation,these data strongly implicate these factors in causing osteoblast anergyand contributing to multiple myeloma bone disease by suppressing thenormal compensatory bone production that follows bone loss.

[0061] The role of multiple myeloma plasma cells in stimulatingosteoclast activity has been intensely investigated and several keylinks established. Data presented herein provide for the first timeevidence of a possible mechanistic explanation of osteoblast dysfunctionin multiple myeloma. These are significant observations in that recentstudies have shown that inhibition of WNT signaling causes defects inosteoblast function. The secreted DKK-1 and FRZB could account for boththe systemic osteoporosis seen in multiple myeloma as well as theexaggerated local bone destruction proximal to plasma cells foci.

[0062] Importantly, DKK-1 and FRZB act to inhibit WNT signaling throughindependent mechanisms, indicating that their co-expression may havesynergistic effects. Thus, these genes could be used to predict extentof bone disease and future risk of developing bone disease. Moreover,inhibitors of these proteins could be used to block bone disease. It isalso possible that these factors play a role in osteoporosis in thegeneral population.

[0063] WNT Signaling Pathway

[0064] Wnt genes comprise a large family of secreted polypeptides thatare expressed in spatially and tissue-restricted patterns duringvertebrate embryonic development. Mutational analysis in mice has shownthe importance of Wnts in controlling diverse developmental processessuch as patterning of the body axis, central nervous system and limbs,and the regulation of inductive events during organogenesis. The Wntfamily of secreted growth factors initiates signaling via the Frizzled(Fz) receptor and its coreceptor, LDL receptor-related protein 5 or 6(LPR5 or LRP6), presumably through Fz-LPR5/LRP6 complex formationinduced by Wnt.

[0065] Secreted antagonists of Wnt include Frizzled (Fz)-relatedproteins (FRPs), Cerberus, Wnt inhibitory factor (WIF) and Dickkopf(DKK). Frizzled (Fz)-related proteins, Cerberus and Wnt inhibitoryfactor have all been shown to act by binding and sequestering Wnt.Unlike Wnt antagonists which exert their effects by molecular mimicry ofFz or Wnt sequestration through other mechanisms, Dickkopf-1 (DKK-1)specifically inhibits canonical Wnt signalling by binding to theLPR5/LRP6 component of the receptor complex.

[0066] DKK-1 is a head inducer secreted from the vertebrate headorganizer and induces anterior development by antagonizing Wntsignaling. DKK-1 is a high-affinity ligand for LRP6 and inhibits Wntsignaling by preventing Fz-LRP6 complex formation induced by Wnt. DKK-1binds neither Wnt nor Fz, nor does it affect Wnt-Fz interaction. DKK-1function in head induction and Wnt signaling inhibition strictlycorrelates with its ability to bind LPR5/LRP6 and to disrupt theFz-LPR5/LRP6 association. LPR5/LRP6 function and DKK-1 inhibition appearto be specific for the Wnt/Fz beta-catenin pathway. These findings thusreveal a novel mechanism for Wnt signal modulation.

[0067] WNT Signaling and Osteoblast Differentiation

[0068] Recent studies have shown that the Wnt signaling pathway iscritical for osteoblast differentiation and function. Mice with atargeted disruption in the gene for low-density lipoproteinreceptor-related protein 5 (LRP5) developed a low bone mass phenotype.LRP5 is expressed in osteoblasts and is required for optimal Wntsignaling in osteoblasts. In vivo and in vitro analyses indicated thatthis phenotype becomes evident postnatally, and it was secondary todecreased osteoblast proliferation and function in a Cbfa1-independentmanner.

[0069] In human, mutations in LRP5 cause the autosomal recessivedisorder osteoporosis-pseudoglioma syndrome (OPPG).Osteoporosis-pseudoglioma syndrome carriers have reduced bone mass whencompared to age- and gender-matched controls.

[0070] Importantly, separate and distinct mutations in LRP result in ahigh bone mass phenotype. In contrast to the osteopororsis-psuedogliomamutations, the high bone mass traits are gain of function mutations.Markers of bone resorption were normal in the affected subjects, whereasmarkers of bone formation such as osteocalcin were markedly elevated.Levels of fibronectin, a known target of signaling by Wnt, were alsoelevated. In vitro studies showed that the normal inhibition of Wntsignaling by Dickkopf-1 (DKK-1) was defective in the presence of themutation and that this resulted in increased signaling due to unopposedWnt activity. These findings demonstrated the role of altered LRP5function in high bone mass and point to DKK as a potential target forthe prevention or treatment of osteoporosis.

[0071] WNT Signaling and Bone Disease in Multiple Myeloma

[0072] Indirect evidence of a role of DKK-1 in osteoblast function hasbeen provided by identification of gain of function mutations in LRP-5being linked to a high bone mass phenotype. In addition, targeteddisruption of secreted firzzled-related protein (SFRP-1), a homologue ofFRZB (SFRP-3), leads to decreased osteoblast and osteocyte apoptosis andincreased trabecular bone formation.

[0073] A quantitative trait loci (QTL) influencing bone mass has beenlocalized to the LRP-5 region, suggesting that the population at largehave different risk of developing osteoporosis. It is conceivable thatmultiple myeloma bone disease may be influenced by the combined effectsof DKK-1/FRZB expression with an inherited predisposition to low bonemass conferred by inherited LRP-5 alleles. Multiple myeloma cases may begenotyped for LRP-5 allele variations and correlate this informationwith bone disease, and DKK-1 and FRZB expression.

[0074] Monoclonal gammopathy of undetermined significance (MGUS), aplasma cell dyscrasia that is predisposed to develop into multiplemyeloma, is differentiated from multiple myeloma by the lack of obviousbone disease. The significance of discovering DKK-1 and/or FRZBexpression in a third of monoclonal gammopathy of undeterminedsignificance is unclear but could suggest that these cases may be athigher risk for developing multiple myeloma. As with multiple myeloma,this predisposition may also be related to inherited LRP5 alleles.Alternatively, these monoclonal gammopathy of undetermined significancecases could have underlying preclinical bone disease that is not yetapparent by radiological scans.

[0075] Data presented herein suggests a model for how DKK-1 expressionby multiple myeloma plasma cells can be linked to multiple myelomadisease growth control and bone destruction and how these two phenomenacan be integrated by one molecule. In the model, primary multiplemyeloma express high levels of DKK and these levels can be increasedwith drug therapies used to treat the disease. High levels of DKK-1likely induce apoptosis of multiple myeloma cells and could explain therelatively slow progression of the disease in its early phase as cellgrowth is tempered by high rate of DKK-1 induced apoptosis. However, asthe disease progresses there is an osteoclast-induced reduction in JUNand DKK-1 that eventually develops into a constitutive loss of JUN andDKK-1 expression as seen in extramedullary disease.

[0076] Thus, if one were to view DKK-1 expression from the perspectiveof the multiple myeloma plasma cells, high levels of DKK-1 expressioncould be seen as positive feature of the disease. However, with themesenchymal cell lineage being exquisitely sensitive to DKK-1 inducedapoptosis, the high levels of this secreted product likely has a doubleedge to it in that it also induces massive programmed cell death ofosteoblast precursors and possibly even mesenchymal stem cells. It isexpected that high levels of DKK-1 early in the disease could lead to apermanent loss of mesenchymal stem cells, a notion supported by theobserved lack of bone repair after remission induction or during diseaseprogression when osteoclasts likely suppress DKK-1 secretion by multiplemyeloma plasma cells. Thus, exploitation of this knowledge might lead tothe development of new therapies for multiple myeloma that accentuateDKK-1's effects on multiple myeloma plasma cells, but at the same timeprevent DKK's bone damaging effects on osteoblast or their precursors.

[0077] In one embodiment of the present invention, there is provided amethod of determining the potential of developing a bone disease in amultiple myeloma patient by examining the expression level of WNTsiganling antagonist. Increased expression of the antagonist compared tothat in normal individual would indicate that the patient has thepotential of developing bone disease. Preferably, the WNT signalingantagonist is soluble frizzled related protein 3 (SFRP-3/FRZB) or thehuman homologue of Dickkopf-1 (DKK1). In general, the expression levelsof these proteins can be determined at the nucleic acid or proteinlevel.

[0078] In another embodiment, there is provided a method of treatingbone disease in a multiple myeloma patient by inhibiting the expressionof WNT signaling antagonist. Preferably, the WNT signaling antagonist issoluble frizzled related protein 3 (SFRP-3/FRZB) or the human homologueof Dickkopf-1 (DKK1). In general, the expression of these antagonistscan be inhibited at the nucleic acid or protein level.

[0079] In yet another embodiment, there is provided a method ofpreventing bone loss in an individual by inhibiting the expression ofWNT signaling antagonist. Preferably, the WNT signaling antagonist issoluble frizzled related protein 3 (SFRP-3/FRZB) or the human homologueof Dickkopf-1 (DKK1). In general, the expression of these antagonistscan be inhibited at the nucleic acid or protein level.

[0080] In yet another embodiment, there is provided a method ofcontrolling bone loss in an individual, comprising the step ofinhibiting the expression of the DKK1 gene (accession number NM012242)or the activity of the protein expressed by the DKK1 gene. The DKK1 geneexpression is inhibited by any method known to a person having ordinaryskill in this art including, e.g., anti-sense oligonucleotides or byanti-DKK1 antibodies or soluble LRP receptors.

[0081] In yet another embodiment, there is provided a method ofcontrolling bone loss in an individual, comprising the step ofadministering to said individual a pharmacological inhibitor of DKK1protein. Generally, this method would be useful where the individual hasa disease such as multiple myeloma, osteoporosis, post-menopausalosteoporosis or malignancy-related bone loss. Generally, themalignancy-related bone loss is caused by breast cancer metastasis tothe bone or prostate cancer metastasis to the bone.

[0082] The following examples are given for the purpose of illustratingvarious embodiments of the invention and are not meant to limit thepresent invention in any fashion. One skilled in the art will appreciatereadily that the present invention is well adapted to carry out theobjects and obtain the ends and advantages mentioned, as well as thoseobjects, ends and advantages inherent herein. Changes therein and otheruses which are encompassed within the spirit of the invention as definedby the scope of the claims will occur to those skilled in the art.

EXAMPLE 1

[0083] Patients

[0084] 174 patients with newly diagnosed multiple myeloma, 16 patientswith monoclonal gammopathy of undetermined significance, 9 withWaldenström's macroglobulinemia, and 45 normal persons were studied. TheInstitutional Review Board of the University of Arkansas for MedicalSciences approved the research studies and all subjects provided writteninformed consent. Table 1 shows the characteristics of the patients withmultiple myeloma. TABLE 1 Myeloma patient characteristics and theirrelationship to MRI lesions P Variable n/N % MRI = 1+ MRI = 0 value Age≧ 65 yr  23/169 14  17/132  6/36 0.59* (12.9%) (16.7%) Caucasian 147/16987 113/132 33/36 0.42* (85.6%) (91.7%) Female  68/169 40  55/132 13/360.55 (41.7%) (36.1%) Kappa light 104/165 63  79/128 24/36 0.59 chain(61.7%) (66.7%) Lambda light  61/165 37  49/128 12/36 0.59 chain (38.3%)(33.3%) IgA subtype  39/169 23  25/132 14/36 0.012 (18.9%) (38.9%) B2M ≧4 mg/L  60/169 36  47/132 13/36 0.96 (35.6%) (36.1%) CRP ≧ 4 mg/L 12/166  7  11/129 (8.5%)  1/36 (2.8%) 0.47* Creatinine ≧  19/169 11 16/132  3/36 (8.3%) 0.77* 2 mg/dL (12.1%) LDH ≧ 190 UI/L  52/169 31 44/132  8/36 0.20 (33.3%) (22.2%) Albumin < 3.5 g/dL  23/169 14  19/132 4/36 0.79* (14.4%) (11.1%) Hgb < 10 g/dL  40/169 24  31/132  8/36 0.87(23.5%) (22.2%) PCLl ≧ 1%  23/150 15  18/119  4/30 1.00* (15.1%) (13.3%)ASPC ≧ 33% 109/166 66  82/129 26/36 0.33 (63.6%) (72.2%) BMPC ≧ 33%104/166 63  79/129 24/36 0.55 (61.2%) (66.7%) Cytogenetic  52/156 33 45/121  6/34 0.032 abnormalities (37.2%) (17.6%) CA13 or  33/52 63 31/121  3/34 (8.8%) 0.037 hypodiploid (25.6%) Other CA  19/52 37 53/103 16/32 0.89 (51.5%) (50.0%) FISH13  69/136 51 103/136 28/36 0.80(75.7%) (77.8%) Osteopenia 131/173 76 1+ Lesions 137/173 79 by MRI 3+Lesions 108/173 62 by MRI 1+ Lesions 105/174 60 by X-ray 3+ Lesions 69/174 40 by X-ray

EXAMPLE 2

[0085] Bone Imaging

[0086] Images were reviewed, without prior knowledge of gene expressiondata, using a Canon PACS (Picture Archiving and Cataloging System). MRIscans were performed on 1.5 Tesla GE Signa™ scanners. X-rays weredigitized from film in accordance with American College of Radiologystandards. MRI scans and x-rays were linked to the Canon PACS systemusing the ACR's DICOM (Digital Imaging and Communications in Medicine)standard. Imaging was done in accordance with manufacturers'specifications. MRI images were created with pre- and post-gadoliniumT1-weighting and STIR (short-tau inversion recovery) weighting.

EXAMPLE 3

[0087] Plasma Cell Isolation and Gene Expression Profiling

[0088] Following Ficoll-Hypaque gradient centrifugation, plasma cellsobtained from the bone marrow were isolated from the mononuclear cellfraction by immunomagnetic bead selection using a monoclonal mouseanti-human CD138 antibody (Miltenyi-Biotec, Auburn, Calif.). More than90 percent of the cells used for gene expression profiling were plasmacells, as shown by two-color flow cytometry using CD138⁺/CD45⁻ andCD38⁺/CD45⁻ markers, the presence of cytoplasmic immunoglobulin lightchains by immunocytochemistry, and morphology by Wright-Giemsa staining.Total RNA was isolated with RNeasy Mini Kit (Qiagen, Valencia, Calif.).Preparation of labeled cRNA and hybridization to U95Av2 microarrayscontaining approximately 10,000 genes (Affymetrix, Santa Clara, Calif.)was performed as previously described (Zhan et al., 2002; Zhan et al.,2003). RNA amplification was not required.

EXAMPLE 4

[0089] Immunohistochemistry

[0090] An antibody from a goat that was immunized against the entirehuman DKK1 protein (R&D Systems, Minneapolis, Minn.) was diluted 1:200in Tris-buffer and added to formalin-fixed, paraffin-embedded bonemarrow biopsy sections for 2 hours at room temperature. Adjacentsections were stained with H & E. Antigen-antibody reactions weredeveloped with DAB (after biotinylated anti-goat antibody [VectorLaboratories, Burlingame, Calif.] [1:400 dilution] andstreptavidin-horse radish peroxidase [Dako] staining), andcounterstained with Hematoxylin-2.

EXAMPLE 5

[0091] Enzyme Linked Immunosorbent Assay (ELISA)

[0092] Nunc-Immuno MaxiSorp surface microtiter plates were coated with50 ml of anti-DKK1 antibody at 1 mg/ml in 1× phosphate buffered saline,pH 7.2 at 4° C. overnight, and blocked with 4 percent bovine serumalbumin. Bone marrow plasma was diluted 1:50 in dilution buffer (1×phosphate buffered saline +0.1 Tween-20+1 percent bovine serum albumin).A total of 50 μl was loaded per well and incubated overnight at 4° C.,washed and incubated with biotinylated goat anti-human DKK1 IgG (R&DSystems) diluted to 0.2 mg/ml in dilution buffer, followed by additionof 50 μl of 1:10,000 dilution of streptavidin-horse radish peroxidase(Vector Laboratories), all according to manufacturer's recommendations.Color development was achieved with the OPD substrate system (Dako)based on manufacturer's instructions. Serial dilutions of recombinanthuman DKK1 (R&D Systems) were used to establish a standard curve. Thecell line T293, which does not express endogenous DKK1 and T293 withstably transfected DKK1 (Fedi, et al., 1999) were used to validate theELISA assay.

EXAMPLE 6

[0093] Osteoblast Differentiation Assays

[0094] C2C12 mesenchymal precursor cells (American Type Tissue Culture,Reston, Va.) were cultured in DMEM (Invitrogen, Carlsbad, Calif.)supplemented with 10 percent heat-inactivated fetal calf serum. Alkalinephosphatase activity in C2C12 cells was measured as described (Gallea,et al., 2001; Spinella-Jaegle, et al., 2001). Cell lysates were analyzedfor protein content using the micro-BCA assay kit (Pierce, Rockford,Ill.). Each experiment was done in triplicate.

EXAMPLE 7

[0095] Statistical Analyses

[0096] Bone disease in multiple myeloma patients was modeled usinglogistic regression. Independent variables considered were geneexpression intensity values (average difference calls) from ˜10,000genes (12,625 probe sets) measured using version 5.01 MAS (Affymetrix,Santa Clara, Calif.) from 174 cases of newly diagnosed multiple myeloma.The “Signal”, a quantitative measure of gene expression, for each probeset was transformed to log₂ before entry into the logistic regressionmodel and permutation-adjustment analysis. There was no prior hypothesiswith regard to genes that might be associated with bone disease inmyeloma. As a result a univariate model of bone disease for each of the12,625 probe sets was used. Candidate genes were refined using t-testswith permutation-adjusted significance levels (Westfall and Young,1993). The Westfall and Young analysis was used to adjust for themultiple univariate hypothesis tests. Group differences in DKK1 signaland DKK1 protein levels were tested using the Wilcoxon rank sum test.Significant differences in patient characteristics by status of bonedisease were tested using either the Fisher's exact test or thechi-square test. Expression intensities of genes identified by logisticregression were visualized with Clusterview (Golub, et. al., 1999).Spearman's correlation coefficient was used to measure correlation ofgene expression and protein levels. Significant differences, inosteoblast differentiation, between the control and each experimentalcondition were tested using the Wilcoxon rank sum test; separatecomparisons were made for each unique C2C12 experiment. Two-sidedp-values less than 0.05 were considered significant and two-sidedp-values less than 0.10 were considered marginally significant.

EXAMPLE 8

[0097] Gene Expression Profiling of Myeloma Cells

[0098] To identify genes that were overexpressed and associated with thepresence of bone lesions, comparing microarray data from patients withor without bone lesions were performed. As MRI-defined focal lesions ofbone can occur before radiologically identifiable lytic lesions,T1-weighted and STIR-weighted imaging to evaluate bone lesions wereused. The gene expression patterns of approximately 10,000 genes inpurified plasma cells from the marrow of patients with no bone lesions(n=36) and those with 1 or more (1+) MRI-defined focal lesions (n=137)were modeled by logistic regression analysis. The model identified 57genes that were expressed differently (P<0.0001) in the two groups ofpatients (FIG. 1A). These 57 genes were further analyzed by t-tests withpermutation-adjusted significance (Westfall and Young, 1993). Thesestatistical tests showed that 4 of the 57 genes were overexpressed inpatients with 1+MRI lesions: dihydrofolate reductase (DHFR), proteasomeactivator subunit (PSME2), CDC28 protein kinase 2 (CKS2), and dickkopfhomolog 1 (DKK1). Given that the gene for the Wnt/β-catenin signalingantagonist DKK1 is the only one of the four that codes for a secretedfactor and that Wnt/β-catenin signaling is implicated in bone biology,further tests on DKK1 were carried out. An analysis of the results fromthe 173 patients with myeloma showed that DKK1 signal for patients with1+MRI and no x-ray lesions differ significantly compared to patientswith no MRI and no x-ray lesions (median signal: 2,220 vs. 285; p<0.001)but does not differ significantly compared to patients with 1+MRI and1+x-ray (median signal: 2,220 vs. 1,865; p=0.63) (FIG. 1B, Table 2).

[0099] Monoclonal gammopathy of undetermined significance (MGUS) is aplasma cell dyscrasia without lytic bone lesions and can precedemultiple myeloma. In 15 of 16 cases of MGUS, DKK1 was expressed by bonemarrow plasma cells at levels comparable to those in multiple myelomawith no MRI or x-ray lesions of bone (FIG. 1B). DKK1 was undetectable inplasma cells from 45 normal donors, and 9 patients with Waldenström'smacroglobulinemia a plasma cell malignancy of the bone lacking bonelesions (FIG. 1B). TABLE 2 DKK1 mRNA and protein levels inMRI/X-ray-lesion defined subgroups of MM No MRI/ 1 + MRI/ 1 + MRI/ NoX-ray No X-ray 1 + X-ray N 36 33 104 DKK1 Mean 536.1 3146.5 3415.1(Signal) (Std) (720.7) (3079.9) (4870.8) (mRNA) DKK1 Min, 19.2, 16.4,9.4, 1864.7, (Signal) Median, 284.9, 2220.2, 28859.1 (protein) Max3810.2 10828.4 N 18 9 41 DKK1 Mean 9.0 (4.7) 24.0 (17.7) 34.3 (75.3)(ng/ml) (Std) (mRNA) DKK1 Min, 1.8, 8.7, 7.4, 20.4, 2.5, 13.5, (ng/ml)Median, 19.7 61.8 475.8 (protein) Max

EXAMPLE 9

[0100] Global Gene Expression Reveals DKK-1 and FRZB Linked to LyticBone Lesion in Multiple Myeloma

[0101] In order to further identify the molecular determinants of lyticbone disease, the expression profiles of ˜12,000 genes in CD138-enrichedplasma cells from newly diagnosed multiple myeloma patients exhibitingno radiological evidence of lytic lesions on bone surveys (n=28) werecompared to those with ≧3 lytic lesions (n=47). The Chi-square test ofabsolute calls (a qualitative measure of gene expression) was used toidentify 30 genes that distinguished the two forms of disease (P<0.05).The Wilcoxon Rank Sum (WRS) test of the signal call (a quantitativemeasure of gene expression) revealed that 104 genes (49 up- and 55down-regulated) differentiated the two disease subtypes (P<0.001).

[0102] The Chi-square test identified the RHAMM proto-oncogene as themost significant discriminator between the two groups. It was expressedin only 7 of 28 patients with no bone disease compared with 34 of 47patients with bone disease (FIG. 2). As expected, plasma cells from only1 of 11 monoclonal gammopathy of undetermined significance expressedRHAMM (FIG. 3). WRS ranked RHAMM as the 14^(th) most significantdiscriminator between the lytic lesion group and no lytic lesion group.NCALD, a calcium binding protein involved in neuronal signaltransduction, was present in 11/28 (40%) of no lytic lesion group butonly in 2/47 (4%) lytic lesion group. Other notable genes identified byChi-square analysis included FRZB, an antagonist of Wnt signaling, thatwas present in 40/47 (85%) of lytic lesion group and 15/28 (53%) of nolytic lesion group. CBFA2/AML1B has been linked to MIP1α expression andwas present in 50% of the no lytic lesion group and in 79% of the lyticlesion group.

[0103] PTTG1 (securin) involved in chromosome segregation was identifiedby WRS as the most significant discriminating gene (P=4×10⁻⁶). It wascalled present in 11% of no lytic lesion group but present in 50% of thelytic lesion group (FIG. 4). Other notable genes in the WRS testincluded the TSC-22 homologue DSIPI which was expressed at lower levelsin lytic lesion group (P=3×10⁻⁵). DSIPI is also down-regulated in 12 of12 multiple myeloma plasma cells after ex-vivo co-culture withosteoclasts.

[0104] In addition, 4 so called “spike genes” were identified that weremore frequently found in lytic lesion group versus no lytic lesion group(p<0.05): IL6, showing spikes in 0/28 no lytic lesion group and 7/47lytic lesion group (p=0.032); Osteonidogen (NID2) showing spikes in 0/28no lytic lesion group and 7/47 lytic lesion group (p=0.032); Regulatorof G protein signaling (RGS13) showing spikes in 1/28 no lytic lesiongroup and 11/47 lytic lesion group (p=0.023); and pyromidinergicreceptor P2Y (P2RY6) showing spikes in 1/28 no lytic lesion group and1/47 lytic lesion group (p=0.023).

[0105] Thus, these data suggest that gene expression patterns may belinked to bone disease. In addition to being potentially useful aspredictors of the emergence of lytic bone disease and conversion frommonoclonal gammopathy of undetermined significance to overt multiplemyeloma, they may also identify targets for potential intervention.

EXAMPLE 10

[0106] DDK1 and FRZB Tend to Be Expressed at Higher Levels in PlasmaCells From Focal Lesions Than From Random Marrow

[0107] Given the relationship of DKK-1 and FRZB to lytic lesions, DKK-1and FRZB expressions were compared in plasma cells derived from randombone marrow aspirates of the iliac crest with those derived by CT-guidedfine needle aspiration of focal lesions of the spine. These resultsshowed significantly higher levels of expression in plasma cells fromfocal lesions.

EXAMPLE 11

[0108] DKK-1 and FRZB Are Not Expressed in Plasma Cells FromWaldenstrom's Macroglobulinemia

[0109] Waldenstrom's macroglobulinemia is a rare plasma cell dyscrasiacharacterized by a monoclonal IgM paraproteinemia and lymphoplasmacyticinfiltration of bone marrow, lymph nodes and spleen. Its clinicalpresentation is quite variable as is the clinical course, yet unlikemultiple myeloma, bone lesions are rare. Although global gene expressionprofiling of CD138-enriched bone marrow plasma cells from 10 cases ofWaldenstrom's Macroglobulinemia reveled gross abnormalities (Zhan etal., 2002), these cells, like normal bone marrow plasma cells, lackexpression of FRZB and DKK (FIG. 20).

EXAMPLE 12

[0110] FRZB and Endothelin Receptor B Are Correlated With DKK-1

[0111] Endothelin 1 is a 21 amino acids vasoconstrictor. Two receptorsfor endothelin, receptors A and B, have been identified. Breast andprostate cancer cells can produce endothelin 1, and increasedconcentrations of endothelin 1 and endothelin receptor A have been foundin advanced prostate cancer with bone metastases. Breast cancer cellsthat produced endothelin 1 caused osteoblastic metastases in femalemice. Conditioned media and exogenous endothelin 1 stimulatedosteoblasts proliferation and new bone formation in mouse calvariaecultures (FIG. 31). These results suggest that endothelin is linked tobone formation.

[0112] Table 3 shows that the expression of endothelin receptor B(ENDRB) was correlated with that of DKK-1. Endothelin receptor B was a‘spike’ gene in one third of newly diagnosed multiple myeloma (FIG. 29).Endothelin receptor B was also expressed in subsets of monoclonalgammopathy of undetermined significance (MGUS) and smoldering multiplemyeloma but not in normal plasma cells (FIG. 30). TABLE 3 CorrelationBetween Endothelin Receptor B (EDNRB) and DKK-1 Gene Symbol Asymp.Significance (two-tailed) DKK-1 6.35 × 10⁻¹⁴ FRZB 6.59 × 10⁻⁸   EDNRB0.00014 DKFZP564G202 4.83 × 10⁻¹¹ IFI27 1.43 × 10⁻⁶   SLC13A3 0.00011CCND1 0.00010 SYN47 4.27 × 10⁻¹⁰ PCDH9 0.00029

EXAMPLE 13

[0113] In Vivo Drug Treatment Upregulates DKK-1

[0114] It has been shown that DKK-1 expression is massively upregulatedby UV irradiation and several other gentoxic stimuli. To see if multiplemyeloma plasma cells also upregulate the genes in response to drugs usedto treat this disease, gene expression profiling of multiple myelomaplasma cells was performed before and after 48 hour in vivo treatmentwith thalidomide (FIG. 33), ImiD (FIG. 34), PS-341 (FIG. 32), ordexamethasone (FIG. 35). These data showed that DKK-1 and FRZBexpression could be massively upregulated in many cases and thussupporting a direct role of DKK-1 in triggering apoptosis of multiplemyeloma plasma cells. It is interesting to note that a newly diagnosedpatient who was primary refractory to all agents tested showed lowlevels of DKK-1 in initial prestudy tests and never showed increasedexpression of DKK-1 or FRZB after drug treatment, supporting a role forDKK-1 expression in promoting apoptosis of multiple myeloma plasmacells. In support of this notion, DKK-1 and FRZB were expressed at lowto undetectable levels in 30 HMCL and several cases of extramedullarydisease (FIG. 15).

EXAMPLE 14

[0115] Co-Culture of Multiple Myeloma with Osteoclasts Results inMassive Downregulation of JUN, FOS, and DKK-1

[0116] The close relationship between myeloma cells and osteoclasts isexpressed clinically by the association of debilitating lytic bonedestruction with multiple myeloma. The development of lytic bone lesionsis caused by the activation of osteoclasts through direct and indirectinteractions with myeloma plasma cells. The critical role of osteoclastsin the survival and growth of myeloma cells and in sustaining thedisease process has been gleaned clinically and demonstrated in vivo inexperimental models such as the SCID-hu model for primary human myeloma.

[0117] In order to investigate the molecular consequences of multiplemyeloma plasma cell/osteoclast interactions, an ex vivo system wasdeveloped in which CD138-enriched multiple myeloma plasma cells wereco-cultured with osteoclasts derived from multiple myeloma peripheralblood stem cells or PBSCs and MNC from healthy donors. CD138-enrichedmultiple myeloma plasma cells co-cultured with human osteoclasts derivedfrom peripheral blood stem cells from normal donors or multiple myelomapatients maintained their viability and proliferative activity asindicated by annexin V flow cytometry, BrdU labeling index and [³H]TdRincorporation for as long as 50 days. Purity level of plasma cellsbefore and after co-cultures was greater than 95% as determined byCD38/CD45 flow cytometry.

[0118] Microarray analyses of the expression of ˜12,000 genes in 12multiple myeloma plasma cells were performed before and after 4 dayco-culture. Heirarchical cluster analysis of the 12 multiple myelomaplasma cells pairs and 150 newly diagnosed multiple myeloma plasma cellsusing 7,913 probes sets (genes) revealed that whereas the pre-co-culturesamples were distributed amongst 3 major cluster groups, thepost-co-culture samples clustered tightly together in 2 of the majorbranches. An analysis of the significant gene expression changes afterco-culture showed that 95 probe sets (genes) changed 2- to 50-fold (77up- and 18 down-regulated) in at least 8 of the 12 multiple myelomaplasma cells after co-culture. CD138-enriched plasma cells from 5healthy donors showed identical shifts in many of the same genes,suggesting that multiple myeloma plasma cells do not exhibit alteredresponses to osteoclasts. However, normal plasma cells as opposed totheir malignant counterparts did not survive in long term co-cultureswith osteoclasts.

[0119] The most striking changes were in the up-regulation of thechemokines GRO1, GRO2, GRO3, SCYA2, SCYA8, SCYA18, and IL8. Othernotable genes included the chemokine receptor CCR1, osteopontin (SPP1),the integrins ITGB2 and ITGB5, matrix metalloproteinase 9 (MMP9),cathepsin K (CTSK) and cathepsin L (CTSL). Surprisingly, a large numberof osteoclast-related genes were among the 77 up-regulated genes. Thedown-regulated genes included cyclin B (CCNB1), the cyclin B specificubiquitin ligase UBE2C, the TSC-22 homologue DSIPI, and JUN, JUND, FOS,and FOSB.

[0120] Gene expression changes were also tested in 10 osteoclastcultured alone and after co-culture with multiple myeloma plasma cells.Twenty-four genes (14 up- and 10 down-regulated) changed 2- to 10-foldin at least 7 of 10 osteoclasts after co-culture. There were nosignificant differences in gene expression between multiple myelomaplasma cells cultured with osteoclasts derived from multiple myelomapatients or from healthy donors, suggesting that multiple myelomaosteoclasts are not qualitatively different than those derived fromnormal donors.

[0121] No significant changes in gene expression were observed whenmultiple myeloma plasma cells were cultured in media derived from aco-culture experiment, suggesting that contact is important. Given thelow ratio of multiple myeloma plasma cells to osteoclasts in theco-culture experiments (1000:1), it is unlikely that all plasma cellscan be in contact with the osteoclasts simultaneously. Thus, it islikely that some intercellular communication between multiple myelomaplasma cells in contact with osteoclasts and those other multiplemyeloma plasma cells occurs.

[0122] It is known that osteoclasts play a major role in multiplemyeloma bone disease as well as providing multiple myeloma withanti-apoptotic signals. Recent studies have shown that JUN directlyregulates DKK-1 expression and that JUN and DKK-1 control apoptosis.

[0123] To determine if osteoclasts may prevent apoptosis of multiplemyeloma plasma cells by modulating JUN and DKK-1, gene expressionprofiling was performed on purified plasma cells from 12 primarymultiple myeloma cases before and after 48 hours of co-culture with invitro derived osteoclasts. Multiple myeloma plasma cells in theco-culture had significantly higher long-term viability than cellscultured alone. Gene expression profiling of multiple myeloma plasmacells before and after osteoclast co-culture revealed that JUN, FOS, andFOSB were 3 of 40 genes down-regulated more than 2-fold in all cases(n=12/12). Hierarchical cluster analysis of HMCL and primary multiplemyeloma cells with 95 genes significantly modulated in multiple myelomaplasma cells after co-culture revealed a striking similarity betweenHMCL, primary multiple myeloma co-cultured with osteoclasts and a subsetof newly diagnosed multiple myeloma in that these cell types hadrelatively low levels of c-JUN and c-FOS.

[0124] Importantly, whereas primary multiple myeloma cells show a highdegree of spontaneous apoptosis when cultured alone, multiple myelomaplasma cells cultured in the presence of osteoclasts can surviveindefinitely. These data support a link between JUN and DKK-1 and alsosuggest that loss of JUN and DKK expression in multiple myeloma may beassociated with disease progression as extramedulalary diseasse andHMCL, which are invariably derived from extramedullary disease, lackboth JUN and DKK. It is interesting to speculate that one of the majorinfluences of osteoclasts on multiple myeloma growth and behavior is todownregulate JUN and DKK-1, which directly affects plasma cellsapoptosis. Treatment of HMCL and primary multiple myeloma/osteoclastsco-cultures with DKK-1 is expected to result in apoptosis of multiplemyeloma plasma cells. DKK-1 will likely have no effect on theosteoclasts, as these cells do not express the Wnt co-receptor LRP-5.Normal bone marrow derived plasma cells also do not express DKK-1 andmay help explain their long-lived nature.

EXAMPLE 15

[0125] Synthesis of DKK1 Protein by Plasma Cells

[0126] Serial sections from bone marrow biopsies of 65 cases of multiplemyeloma were stained for the presence of DKK1. The plasma cells in thesecases contained DKK1 in a manner consistent with the gene expressiondata (FIG. 39). Similar experiments with biopsies from 5 normal donorsfailed to identify DKK1 in any cell. There was a strong tendency forDKK1 positive myelomas to have low-grade morphology (abundant cytoplasmwithout apparent nucleoli) with an interstitial growth pattern. Thisstaining was found to be greatest in plasma cells adjacent to bone. DKK1negative myelomas tend to bear high-grade morphology (enlarged nucleiand prominent nucleoli) with a nodular or obliterative growth pattern.In biospies with an interstitial growth pattern, DKK1 was either present(in varying percentages of cells) or absent. In contrast, myelomas withthe more aggressive nodular growth patterns DKK1 was uniformly absent.Importantly, in cases with both interstitial and nodular growth, theinterstitial cells were positive and the nodular cells negative.

EXAMPLE 16

[0127] DKK1 Protein in Bone Marrow Plasma

[0128] An enzyme-linked immunosorbent assay (ELISA) showed that theconcentration of DKK1 protein in the bone marrow plasma from 107 of the173 newly diagnosed multiple myeloma patients for which gene expressiondata was also available, was 24.02 ng/ml (S.D. 49.58). In contrast, DKK1was 8.9 ng/ml (S.D. 4.2) in 14 normal healthy donors, 7.5 ng/ml (S.D.4.5) in 14 cases of MGUS, and 5.5 ng/ml (S.D. 2.4) in 9 cases ofWaldenström's macroglobulinemia. DKK1 gene expression and the level ofDKK1 in the bone marrow plasma were positively correlated (r=0.65,P<0.001) in the 107 cases of myeloma (FIG. 40A). There was also a strongcorrelation between DKK1 protein levels in bone marrow plasma andperipheral blood plasma in 41 cases of myeloma in which both sampleswere taken simultaneously (r=0.57, P<0.001).

[0129] In 68 patients in whom both DKK1 protein levels in the bonemarrow plasma and the presence of bone lesions were determined, DKK1protein in patients with 1+MRI and no x-ray lesions differ significantlycompared to patients with no MRI and no x-ray lesions (median level: 20ng/ml vs. 9 ng/ml; p=0.002), but does not differ significantly comparedto patients with 1+MRI and 1+x-ray lesions (median level: 20 ng/ml vs.14 ng/ml; p=0.36) (FIG. 40B, Table 2).

EXAMPLE 17

[0130] Effect of Bone Marrow Serum on Osteoblast Differentiation InVitro

[0131] Bone morphogenic protein-2 can induce differentiation of theuncommitted mesenchymal progenitor cell line C2C12 (Katagiri, et al.,1994) into osteoblasts through a mechanism that involves Wnt/b-cateninsignaling (Bain, et al., 2003; Roman-Roman, et al., 2002). Alkalinephosphatase, a specific marker of osteoblast differentiation, wasundetectable in C2C12 cells grown in 5 percent fetal calf serum for 5days (FIG. 41A). Treatment of C2C12 cells with 50 ng/ml of BMP-2 for 5days induced them to produce alkaline phosphatase, whereas alkalinephosphatase was not produced by C2C12 cells that were concomitantlycultured with BMP-2 and 50 ng/ml recombinant human DKK1. This in vitroeffect on alkaline phosphatase production was neutralized by apolyclonal anti-DKK1 antibody, but not by a non-specific polyclonal goatIgG. Bone marrow serum with a DKK1 concentration >12 ng/ml from fivepatients with myeloma inhibited the production of alkaline phosphataseby C2C12 cells treated with BMP-2, and this effect was reversed by theanti-DKK1 antibody, but not by non-specific IgG (FIG. 41B). By contrast,C2C12 cells treated with 50 ng/ml BMP-2 and 10 percent serum from thebone marrow of a normal donor induced the production of alkalinephosphatase by the cells (FIG. 41B).

[0132] The following references were cited herein:

[0133] Zhan et al., Global gene expression profiling of multiplemyeloma, monoclonal gammopathy of undetermined significance, and normalbone marrow plasma cells. Blood 99:1745-1757 (2002).

[0134] Zhan et al., Gene expression profiling of human plasma celldifferentiation and classification of multiple myeloma based onsimilarities to distinct stages of late-stage B-cell development. Blood101:1128-1140 (2003).

[0135] Fedi et al. Isolation and biochemical characterization of thehuman Dkk-1 homologue, a novel inhibitor of mammalian Wnt signaling. JBiol Chem 274:19465-72 (1999).

[0136] Gallea et al. Activation of mitogen-activated protein kinasecascades is involved in regulation of bone morphogeneticprotein-2-induced osteoblast differentiation in pluripotent C2C12 cells.Bone 28:491-8 (2001).

[0137] Spinella-Jaegle et al. Opposite effects of bone morphogeneticprotein-2 and transforming growth factor-beta1 on osteoblastdifferentiation. Bone 29:323-30 (2001).

[0138] Westfall and Young. Resampling-based multiple testing: Examplesand methods for p-value adjustment. Hoboken, N.J.: Wiley-Interscience,360 (1993).

[0139] Golub et al. Molecular classification of cancer: class discoveryand class prediction by gene expression monitoring. Science 286:531-7(1999).

[0140] Katagiri et al. Bone morphogenetic protein-2 converts thedifferentiation pathway of C2C12 myoblasts into the osteoblast lineage.J Cell Biol 127:1755-66 (1994).

[0141] Bain et al. Activated beta-catenin induces osteoblastdifferentiation of C3H10T1/2 cells and participates in BMP2 mediatedsignal transduction. Biochem Biophys Res Commun 301:84-91 (2003).

[0142] Roman-Roman et al. Wnt-mediated signalling via LRP5 andbeta-catenin induce osteoblast differentiation and mediates the effectsof BMP2, American Society of Bone Mineral Research, 2002.

[0143] Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A method of determining the potential ofdeveloping bone disease in a multiple myeloma patient, said methodcomprises the step of: examining the expression level of WNT signalingantagonist, wherein increased expression of said antagonist compared tothat in normal individual indicates that said patient has the potentialof developing bone disease.
 2. The method of claim 1, wherein said WNTsignaling antagonist is soluble frizzled related protein 3 (SFRP-3/FRZB)or the human homologue of Dickkopf-1 (DKK1).
 3. The method of claim 1,wherein said expression level is determined at the nucleic acid level orprotein level.
 4. A method of treating bone disease in a multiplemyeloma patient, said method comprises the step of inhibiting theexpression of a WNT signaling antagonist.
 5. The method of claim 4,wherein said WNT signaling antagonist is soluble frizzled relatedprotein 3 (SFRP-3/FRZB) or the human homologue of Dickkopf-1 (DKK1). 6.The method of claim 4, wherein the expression of said antagonist isinhibited at the nucleic acid level or protein level.
 7. A method ofpreventing bone loss in an individual, said method comprises the step ofinhibiting the expression of a WNT signaling antagonist.
 8. The methodof claim 7, wherein said WNT signaling antagonist is soluble frizzledrelated protein 3 (SFRP-3/FRZB) or the human homologue of Dickkopf-1(DKK1).
 9. The method of claim 7, wherein the expression of saidantagonist is inhibited at the nucleic acid level or protein level. 10.A method of controlling bone loss in an individual, comprising the stepof inhibiting the expression of the DKK1 gene (accession numberNM012242) or the activity of the protein expressed by the DKK1 gene. 11.The method of claim 10, wherein said DKK1 gene expression is inhibitedby anti-sense oligonucleotides, by anti-DKK1 antibodies or soluble LRPreceptors.
 12. A method of controlling bone loss in an individual,comprising the step of administering to said individual apharmacological inhibitor of DKK1 protein.
 13. The method of claim 12,wherein said individual has a disease selected from the group consistingof multiple myeloma, osteoporosis, post-menopausal osteoporosis andmalignancy-related bone loss.
 14. The method of claim 13, wherein saidmalignancy-related bone loss is caused by breast cancer metastasis tothe bone or prostate cancer metastasis to the bone.